Abstract

Hearing loss (HL) is the most common sensory disorder, affecting all age groups, ethnicities, and genders. Several genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Mutations in GJB2 gene, the gene encoding gap junction protein connexin26 (Cx26), are most common detected in patients with congenital, recessively inherited, nonsyndromic HL in humans. In order to investigate the molecular etiology of patients with congenital, recessively inherited, and nonsyndromic HL and healthy individuals as control in a family, they were included in this study. Thus, exons of GJB2 gene were amplified by polymerase chain reaction (PCR) and sequenced. In this family, V27I missense mutation and V27I + E114G compound heterozygosity were detected in the results of sequence analysis. The V27I mutation was found in patient with severe HL and healthy individuals. The V27I + E114G compound heterozygosity was detected only in deaf patients. Based on our data, V27I mutations could be considered as a polymorphism not leading to HL. Since V27I + E114G compound heterozygosity was found only in deaf patients, it could be considered as a contributor of HL severity.

Keywords

GJB2 gene; Hearing loss; Sequencing; Missense mutation

Introduction

Hearing loss is the most common congenital sensory impairment
worldwide [1], which affects approximately 3% of the population [2].
In most of these cases, the inheritance pattern is autosomal recessive
(75-80%), although autosomal dominant (20%), X-linked (2-5%) and
mitochondrial (<1%) inheritance also occur [3]. 30% of cases of
prelingual hearing loss disorders are classified as syndromic, in cases
where additional physical findings lead to HL; the remainders are nonsyndromic
[4].

Hearing loss affects approximately 70 million people worldwide.
Environmental causes, account for 50-60% of cases, can range from neonatal insults, such as prematurity, jaundice, or prenatal infection to
iatrogenic causes. The genetic etiologies, account for 40-50% of cases,
can be inherited as either syndromic or non-syndromic forms, or have
a spectrum of inheritance patterns [2,5]. Non-syndromic deafness
accounts for 60-70% of inherited hearing impairment and involves
more than 100 different genes with autosomal dominant (DFNA),
autosomal recessive (DNFB), X-linked (DFN), and maternal
inheritance. The most common cause of non-syndromic autosomal
recessive hearing loss is mutations in Cx26, a gap junction protein
encoded by the GJB2gene which is located at chromosome 13 q11-12
[6]. Expression of GJB2 has been well documented in a variety of cells
and tissues. In the cochlea, it is believed that GJB2 plays a critical role
in the recycling of K+ by ferrying K+ away from the hair cells during
auditory transduction [7].

Autosomal recessive nonsyndromic sensorineural hearing
impairment (ARNSHI) comprises 80% of familial HL cases. Mutations
in the GJB2, the gene encoding gap junction protein Cx26, are the
largest genetic etiologic contributor to ARNSHI [7]. To date, more
than 100 mutations either leading or contributing to HL have been reported in literature. The most common mutation in Europe, North
America, and the Mediterranean is a deletion of 6 guanine nucleotides
referred to as 35delG [2]. The V37I and V27I are other GJB2 variants,
which are much discussed in literature and usually considered as a
genetic risk-indicator of HL and a determinant of severity of disease
[8].

It has been well documented that GJB2 mutations diverge largely
among different ethnic groups. 35delG is commonly seen in
Caucasians [4], although 167delT has high prevalence in the
Ashkenazi Jewish population [9]. This present study was performed to
determine GJB2 gene variants of a Turkish family with autosomal
recessive nonsyndromic congenital HL.

Materials and Methods

Subjects and molecular genetic analysis

A familial HL patients with autosomal recessive nonsyndromic
congenital HL and their healthy family members were included in this
study if the following criteria were met

(1) There exist hearing-impaired siblings.

(2) There exist healthy control sibling at least one.

(3) There is no evidence of any obvious syndrome. The family with
hearing loss consists of 4 deaf patients and 2 healthy persons.

Results

A familial HL case showing autosomal recessive non-syndromic
sensorineural hearing impairment was included in present study as a
case report shown in (Figure 1). Their hearing losses were
nonprogressive and were either congenital or prelingual in onset.
Cases’ disease severities are indicated in (Table 1).

Patient and Control
Numbers

Genotype

Disease Severity

II:1

V27I+E114G/ wt

Deaf

II:2

V27I+E114G/ wt

Deaf

II:3

wt / wt

Severe

II:4

V27I/ wt

Severe

II:5

V27I/ wt

Healthy

II:6

V27I+E114G/ wt

Deaf

I:1

V27I/ wt

Healthy

Table 1: Genotype and disease severity in patients and controls.

Figure 1: Pedigree and GJB2 genotypes for the study family with
nonsyndromic recessive HL.

Figure 2: Sequence result of a patient with V27I missense mutation.

Results of GJB2 sequence analysis in the present study showed that
V27I mutation shown in Figure 2 was found in both healthy
individuals and patients with severe HL. Previous studies have
indicated that V27I mutation is usually considered as a noncausative
mutation when it is seen alone [10,11]. The V27I mutation was also detected in a compound heterozygous state with E114G mutation, the
Glu to Gly at amino acid position 114 shown in (Figure 2), in only
completely deaf patients.

In autosomal recessive single gene disorders, the allelic
configurations of detected variants play a key role both in determining
correct molecular diagnosis and in disease treatment [12]. V27I
+E114G heterozygous state in cis markedly devastated the function of
the gap junctions that have crucial role in hearing process and it has
been shown that E114G mutation was always detected on the same
chromosome that carries the V27I variant [13]. So the heterozygous
state found in present study could be considered as contributor to
disease severity (Figure 3).

Figure 3: Sequence result of a patient with E114G missense mutation.

Discussion

To date, over 20 different genes associated with recessive deafness
have been identified. GJB2 gene, which is largely expressed in the
cochlea, is involved in both syndromic and nonsyndromic HL with a
dominant or recessive inheritance pattern. Mutation of the GJB2 gene
is the major contributor to autosomal recessive HL and, to a small
percentage, autosomal dominant HL. The molecular genetic analyses
of the GJB2 gene in a family that consist of both HL patients and
healthy individuals showed various variants depending on the severity
of the disease. As can be seen in (Figure 1) showing the molecular
genetic results of the family, V27I missense mutation in both patients
with mild to severe HL and healthy members was identified, although
V27I+E114G compound heterozygote was detected only in deaf
patients.

The V27I mutation is very often found in patients with HL and it
has been considered as a polymorphism not leading to HL [10,11].
Since V27I, the sequence change of the Val to Ile at amino acid
position 27, was also detected in healthy individuals in the present
study, it cannot be regarded as causative mutation of HL. It might be
thought as a contributor because of being located in transmembrane
protein domain. The V27I mutation was also detected in a compound
heterozygous state with E114G mutation, the Glu to Gly change at
amino acid position 114. In autosomal recessive single gene disorders,
the allelic configurations of detected variants play a key role both in
determining correct molecular diagnosis and in disease treatment.
Depending on whether these changes are on the same (in cis) or on opposite (in trans) chromosomes, they can be considered as a
causative or noncausative [12].

Previous results of in vitro functional studies on the Xenopus
oocyte system demonstrated that [13]. It has been found that the V27I
and E114G variants are appeared very often in both deaf probands and
hearing controls and the E114G variant is always in cis with the V27I
variant. Although in vitro experiments suggest a pathogenic role for
the complex allele, the equal distribution of p.[V27I; E114G] in deaf
probands and hearing controls makes it a less likely cause of profound
congenital deafness [14] Pandya et al. suggested that when these two
variants are shown together in cis-configuration, homozygous V27I
+E114G or compound heterozygote with another mutation could
cause hearing loss [13]. Choi et al. used in-vitro assay and population
study to examine the pathogenesis of V27I and E114G variants and
indicated that E114G variant is deleterious but V27I+E114G represent
a nonpathogenic polymorphism and suggesting that only E114G
homozygote or compound heterozygote carrying E114G type with
other mutations may cause HL [15]. Also, V27I+E114G/wt genotype
was found as the cause of hearing loss in a family involved in a
previous study [16]. Two polymorphisms (V27I and E114G) detected
in GJB2 gene were not pathogenic variants causing inherited deafness
which had been described in a previous study [17]. Moreover, while
p.V27I mutation is not leading to HL, V27I + R75W compound
heterozygous state was reported as a disease causative mutation [18].

Based on our data, V27I missense mutation and V27I + E114G
compound heterozygote detected in GJB2 gene involved in a familial
HL case are nonpathogenic variants. Our study indicates that these
mutations are not the main causative mutations associated with HL in
this family. However, since V27I + E114G compound heterozygous
state in our study was found in only deaf patients, it could be thought
that these variants impair the function of gap junctions and play a role
in increasing the severity of HL. This information will be valuable in
considering HL cases or early diagnosis.